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[F. Others] F-7

Antisense Oligonucleotide (ASO) Conjugated With Cell-

Penetrating Peptide Effectively Reduces Target Gene Via

Direct Penetration And Cell-To-Cell Transfer

Seulmee Shin¹, Youngjin Seo¹, Shinyoung Kang¹, Youlim Hong¹, Wookyung Kang¹, Eunna Chung ¹,

Deawoong Jo¹*

¹Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea

Antisense oligonucleotide (ASO) is one of the RNA-therapeutics based on RNaseH-mediated cleavage and

subsequent degradation of complementary RNAs. In the bench-top research, the endocytic delivery of ASO into a
single-cell level is possible, which is limited for in-vivo ASO delivery in a multicellular and systemic environment,

leading to clinical application. Here, to enhance the systemic delivery of ASO, cell-penetrating peptides (CPPs)
named advanced macromolecule transduction domain (aMTD) have been applied. The knockdown (KD) activity of

ASO conjugated with aMTDs (aMTD-ASO) was evaluated by using qPCR. aMTD-ASO displayed superior KD activity
(~37-fold compared to naked-ASO) on the target gene at 4 hrs, which is an insufficient period for endocytosis. In

the presence of endocytosis inhibitors even for enough time capable of endocytic entry (48 hrs), aMTD-ASO also
showed significant KD activity (~30-fold), indicating that aMTD facilitates the intracellular delivery of ASO via direct

penetration rather than endocytosis. Moreover, neighboring cells that were not directly treated with naked-
ASO/aMTD-ASO also showed KD activity (~63%) on the target gene following a co-culture with the treated cells,

suggesting aMTD-ASO can be delivered via cell-to-cell transfer. In conclusion, aMTD enhances delivery of ASO
through direct penetration and cell-to-cell transfer, suggesting a new practical paradigm of RNA-therapeutics.